Exploiting the Symmetry of the Resonator Mode to Enhance PELDOR Sensitivity

Abstract

Pulsed electron paramagnetic resonance (EPR) spectroscopy using microwaves at two frequencies can be employed to measure distances between pairs of paramagnets separated by up to 10 nm. The method, combined with site-directed mutagenesis, has become increasingly popular in structural biology for both its selectivity and capability of providing information not accessible through more standard methods such as nuclear magnetic resonance and X-ray crystallography. Despite these advantages, EPR distance measurements suffer from poor sensitivity. One contributing factor is technical: since 65 MHz typically separates the pump and detection frequencies, they cannot both be located at the center of the pseudo-Lorentzian microwave resonance of a single-mode resonator. To maximize the inversion efficiency, the pump pulse is usually placed at the center of the resonance, while the observer frequency is placed in the wing, with consequent reduction in sensitivity. Here, we consider an alternative configuration: by spacing pump and observer frequencies symmetrically with respect to the microwave resonance and by increasing the quality factor, valuable improvement in the signal-to-noise ratio can be obtained.

A Gadolinium Spin Label with Both a Narrow Central Transition and Short Tether for Use in Double Electron Electron Resonance Distance Measurements

“…The pump pulse was set on the maximum of the Gd­(III). At Q-band both the pump and observer pulses were offset from the minimum of the overcoupled cavity to maximize their frequency difference while maintaining a reasonable level of available power . Therefore, 14 ns was the shortest pump pulse we could use while retaining 32 ns observer pulse lengths.…”

“…At Q-band both the pump and observer pulses were offset from the minimum of the overcoupled cavity to maximize their frequency difference while maintaining a reasonable level of available power. 55 Therefore, 14 ns was the shortest pump pulse we could use while retaining 32 ns observer pulse lengths. We also found that 14 ns was an optimal length for inverting the echo at the maximum of the Gd(III) spectrum, even at the matched frequency of the cavity (34.0 GHz) where there is sufficient power for shorter pulses.…”

Accurate Extraction of Nanometer Distances in Multimers by Pulse EPR

“…Thus, we decided to interchange the spectral positions in a first instance and also the approximate excitation widths of detection and pump pulses in a second stage. Implications of frequency positions with respect to the resonator mode have been discussed recently 24. While the reduction in Δ is unequivocal, the performance of these experiments based on a (spectrally) minor broad inversion25 shows consistent improvement in distance distributions, but appears to give mixed results in terms of sensitivity.…”

Controlling and exploiting intrinsic unpaired electrons in metalloproteins

“…To maximize inversion of the spins the pump pulse is typically placed in the center of the resonance, however symmetrically placing the pulses about the center can increase sensitivity (Salvadori et al, 2015). As metalloprotein cofactors typically have anisotropic and broad EPR signals, the optimal position of the pump and probe pulses in the EPR spectrum depends on the choice of the components to be excited.…”